Dioxin analogues and methods and kits for searching out dioxins-decomposing organisms or enzymes or dioxin-decomposing enzyme genes

ABSTRACT

The present invention provides a novel dioxin analogue for use in the search for organisms capable of degrading dioxin. The dioxin analogue is represented by the following chemical formula (1) or (2):

This application is a U.S. national stage of International ApplicationNo. PCT/JP01/02627 filed Mar. 29, 2001.

TECHNICAL FIELD

The present invention relates to novel dioxin analogues, methods andkits for screening for organisms or enzymes capable of degradingdioxins.

TECHNICAL BACKGROUND

Dioxins are environmental pollutants that have raised public concernbecause of their toxicity and their ability to remain in the environmentfor a long time. No practical solution has been proposed so far toeffectively eliminate dioxins from the environment. One approach is tocollect dioxin-polluted materials in one place for physical and chemicalprocessing. This approach is considered difficult, however, especiallywhen materials contaminated with low-level dioxins are spread over alarge area. Bioremediation, a process that takes advantage of biologicalactivities of microbes in cleaning the environment, is currentlyconsidered the most effective solution to address such low-levelpollution. In constructing an effective bioremediation system, it iscrucial to isolate naturally occurring organisms that have a strongability to rapidly degrade, metabolize, and detoxify dioxins.

Conventional screening processes for dioxin-degrading organisms involveuse of dioxins themselves or radioactively labeled other compounds inorder to evaluate the ability of organisms to degrade dioxins.

Some of these processes require a pre-treatment in evaluating organisms'capability while others require special instruments to detectradioactively labeled compounds. Also, analytical procedures such as gaschromatography and HPLC employed in these processes are time-consuming.

Accordingly, it is an objective of the present invention to eliminatethe above-described drawbacks of the conventional screening techniquesfor dioxin-degrading organisms by providing a novel screening methodthat permits even more efficient screening for desired organisms. Thepresent invention also provides compounds and a screening kit for use inthis screening process.

DISCLOSURE OF THE INVENTION

In a typical biological breakdown pathway of dioxin utilized by, forexample, dibenzo-p-dioxin-degrading fungi, the rate-limiting step is thecleavage of ether bond in the dibenzo-p-dioxin structure, which providesthe backbone common among dioxins. The reaction process is as follows:

The present inventors have directed their interests to the biologicalbreakdown process of dibenzo-p-dioxin shown above and have found that,by substituting one of the benzene rings of dibenzo-p-dioxin with afluorescent compound having a benzene ring, using this substituteddioxin analogue as a substrate in the same reaction process, and bydetecting fluorescence emitted by metabolites, it can easily bedetermined whether an organism of interest, an enzyme of interest, or agene encoding an enzyme of interest has an ability to degrade dioxin.This finding ultimately led the present inventors to complete thepresent invention.

Accordingly, the present invention provides the following items (1) to(10):

-   (1) A novel dioxin analogue represented by the following chemical    formula (1):

-   (2) A novel dioxin analogue represented by the following chemical    formula (2):

-   (3) A method for screening for an organism or an enzyme capable of    degrading dioxin, or a gene encoding an enzyme capable of degrading    dioxin. The method includes the steps of using a dioxin analogue as    a substrate in a reaction involving an organism of interest, an    enzyme of interest, or a gene encoding an enzyme of interest; and    detecting fluorescence emitted by resulting metabolites. The dioxin    analogue is represented by the following general formula (3):

wherein X represents a chlorine atom, m represents an integer from 1 to4, and R¹ represents a hydrogen atom or a lower alkyl group having 1 to3 carbon atoms.

-   (4) The method according to item (3) above, wherein the dioxin    analogue represented by the general formula (3) is the dioxin    analogue represented by the chemical formula (1):

-   (5) A method for screening for an organism or an enzyme capable of    degrading dioxin, or a gene encoding an enzyme capable of degrading    dioxin. The method includes the steps of using a dioxin analogue as    a substrate in a reaction involving an organism of interest, an    enzyme of interest, or a gene encoding an enzyme of interest; and    detecting fluorescence emitted by resulting metabolites. The dioxin    analogue is represented by the following general formula

wherein X represents a chlorine atom, n represents an integer from 1 to4, and R² represents a hydrogen atom or a lower alkyl group having 1 to3 carbon atoms.

-   (6) The method according to item (5) above, wherein the dioxin    analogue represented by the general formula (4) is the dioxin    analogue represented by the chemical formula (2):

-   (7) A screening kit for an organism or an enzyme capable of    degrading dioxin, or a gene encoding an enzyme capable of degrading    dioxin. The kit includes a dioxin analogue represented by the    following general formula (3):

wherein X represents a chlorine atom, m represents an integer from 1 to4, and R² represents a hydrogen atom or a lower alkyl group having 1 to3 carbon atoms.

-   (8) The method according to item (7) above, wherein the dioxin    analogue represented by the general formula (3) is the dioxin    analogue represented by the chemical formula (1):

-   (9) A screening kit for an organism or an enzyme capable of    degrading dioxin, or a gene encoding an enzyme capable of degrading    dioxin. The kit includes a dioxin analogue represented by the    following general formula (4):

wherein X represents a chlorine atom, n represents an integer from 1 to4, and R² represents a hydrogen atom or a lower alkyl group having 1 to3 carbon atoms.

-   (10) The method according to item (9) above, wherein the dioxin    analogue represented by the general formula (4) is the dioxin    analogue represented by the chemical formula (2):

In the present invention, “dioxins that are degradable by an organism oran enzyme capable of degrading dioxin, or a gene encoding an enzymecapable of degrading dioxin” refer to polychlorodibenzo-p-dioxins,including 2,3,7,8-tetrachlorodibenzo[b,e][1,4]dioxin.

In the present invention, dioxin analogues represented by the chemicalformulae (1) and (2) are each a novel compound.

The novel dioxin analogue of the chemical formula (1) can be obtained,for example, by reacting 4-methylesculetin with1,2,4,5-tetrachlorobenzene in hexamethylphosphoramide (HMPA) solvent inthe presence of sodium hydride and a crown ether (18-Crown-6) underinert gas atmosphere.

The novel dioxin analogue of the chemical formula (2) can be obtained,for example, by reacting 2,3-dihidroxynaphthalene with1,2,4,5-tetrachlorobenzene in hexamethylphosphoramide (HMPA) solvent inthe presence of sodium hydride and a crown ether (18-Crown-6) underinert gas atmosphere.

In essence, the method in accordance with the present invention forscreening for an organism or an enzyme capable of degrading dioxin, or agene encoding an enzyme capable of degrading dioxin, makes it possibleto determine whether an organism of interest, an enzyme of interest, ora protein encoded by a gene of interest, has an ability to degradedioxin. This is done by using, as a substrate, a dioxin analogue of thegeneral formula (3) (which is referred to as an esculetin-type dioxinanalogue, hereinafter) or a dioxin analogue of the general formula (4)(which is referred to as a naphthalene-type dioxin analogue,hereinafter) in a reaction involving the organism, or the enzyme, or thegene of interest and detecting the fluorescence emitted by the resultingmetabolites.

While R¹ in the esculetin-type dioxin analogue of the general formula(3) may be any of lower alkyl groups having 1 to 3 carbon atoms,including methyl, ethyl and propyl, the compound preferably has thestructure shown by the chemical formula (1).

Similarly, while R² in the naphthalene-type dioxin analogue of thegeneral formula (4) may be any of lower alkyl groups having 1 to 3carbon atoms, including methyl, ethyl, and propyl, the compoundpreferably has the structure shown by the chemical formula (2).

As used herein, the phrase “an organism capable of degrading dioxin”refers to an organism that can catabolize dioxin down to carbon dioxideor can detoxify or alter dioxin through its metabolic system orbiological activity.

As used herein, the phrase “an enzyme capable of degrading dioxin”refers to an enzyme that is directly or indirectly involved in thecleavage of the ether bonds in the dioxin structure or is directly orindirectly involved in the dechlorination or hydroxylation ofchlorinated dioxins.

As used herein, the phrase “a gene encoding an enzyme capable ofdegrading dioxin” refers to a gene that codes for the above-describedenzyme or a regulatory protein of the enzyme, or any related gene.

As described above, the method of the present invention for screeningfor an organism or an enzyme capable of degrading dioxin involves usingas a substrate the aforementioned esculetin-type dioxin analogue or thenaphthalene-type dioxin analogue in a reaction involving an organism oran enzyme of interest and detecting the fluorescence emitted by themetabolites resulting from the degradation process.

Since the products resulting from the cleavage of the ether bonds in thedioxane ring of dioxin analogues (shown below as the chemical formulas(1a), (1b), (2a), (2b) and (2c)) emit fluorescence, they can be used asan index to determine whether an organism of interest has the ability todegrade dioxin.

Of the degradation products shown in the chemical reactions above, thosederived from the esculetin-type dioxin analogue (i.e., chemical formulas(1a) and (1b)) emit fluorescence at 450 to 460 nm when exposed toexcitation light of 350 to 380 nm, whereas those derived from thenaphthalene-type dioxin analogue (i.e., chemical formulas (2a), (2b) and(2c)) emit fluorescence at 400 to 450 nm when exposed to excitationlight of 350 to 360 nm. Thus, an organism of interest can be assayed forthe ability to degrade dioxin by irradiating the metabolites, which haveresulted from the reaction in which the esculetin-type dioxin analogueor the naphthalene-type dioxin analogue is used as a substrate, eitherwith excitation light with a wavelength of 350 to 380 nm (foresculetin-type dioxin analogue) or with excitation light with awavelength of 350 to 360 nm (for naphthalene-type dioxin analogue) anddetecting the fluorescence at 450 to 460 nm (for esculetin-type dioxincompound) or at 400 to 450 nm (for naphthalene-type dioxin compound).The fluorescence can be detected by, for example, a spectrophotometer.The fluorescence detected at 450 to 460 nm (for esculetin-type dioxinanalogue) or at 400 to 450 nm (for naphthalene-type dioxin analogue)indicates that the organism of interest has the ability to degradedioxin. Alternatively, the metabolites may be visually observed for theemission of fluorescence to screen for an organism capable of degradingdioxin.

In one method of the present invention for screening for an organismcapable of degrading dioxin, an organism of interest is cultured in aculture medium added with the above-described substrate (esculetin-typedioxin analogue or naphthalene-type dioxin analogue) dissolved in asmall amount of an organic solvent. After culturing for a predeterminedperiod of time, a portion of the supernatant of the culture is collectedand is diluted with an alkaline buffer (100 mM glycine, 100 mM sodiumhydroxide, pH 10). The collected sample is irradiated with excitationlight having a wavelength of 350 to 380 nm (for esculetin-type dioxinanalogue) or 350 to 360 nm (for naphthalene-type dioxin analogue) and,using a fluorescence spectrophotometer, fluorescence is detected at 450to 460 nm (for esculetin-type dioxin analogue) or at 400 to 450 nm (fornaphthalene-type dioxin analogue). Alternatively, the fluorescence maybe detected using a spectrophotometer. The organic solvent is preferablyDMSO, acetone or other water-soluble organic solvents and is preferablyadded to the culture medium at a concentration not exceeding 1%. When itis desired to add a surfactant to the culture medium, the substrate isdissolved in a water-insoluble organic solvent such as nonane or hexaneand is preferably added to the culture medium such that the finalconcentration of the surfactant does not exceed 1%. The medium isthoroughly mixed to disperse the substrate.

In one method of the present invention for screening for an enzymecapable of degrading dioxin, the present substrate dissolved in a buffer(pH 4 to 7) is placed in a glass test tube along with a crude enzyme(crude extract) or a fractionated enzyme obtained from an organismsuspected of having the ability to degrade dioxin, and the reaction isallowed to proceed for a predetermined period of time. Subsequently,some or all of the reaction mixture is transferred to another glass testtube and is diluted with an alkaline buffer (100 mM glycine, 100 mMsodium hydroxide, pH 10). The mixture is then transferred to a silicacell for fluorescence analysis and is irradiated with excitation lighthaving a wavelength of 350 to 380 nm (for esculetin-type dioxinanalogue) or 350 to 360 nm (for naphthalene-type dioxin analogue). Usinga fluorescence spectrophotometer, fluorescence is detected at 450 to 460nm (for esculetin-type dioxin analogue) or at 400 to 450 nm (fornaphthalene-type dioxin analogue). Alternatively, the coloring of thesolution may be detected using a spectrophotometer. The organic solventis preferably DMSO, acetone or other water-soluble organic solvents andis preferably added to the reaction solution at a concentration notexceeding 1%. When it is desired to add a surfactant to the reactionsolution, the substrate is dissolved in a water-insoluble organicsolvent such as nonane or hexane and is preferably added to the culturemedium such that the final concentration of the surfactant does notexceed 1%. The solution is thoroughly mixed to disperse the substrate.

In one method of the present invention for screening for a gene encodingan enzyme capable of degrading dioxin, a gene library of an organismexpressing the ability to degrade dioxin is introduced into a bacterium,such as E. Coli, or a eukaryotic microorganism, such as yeast, which isincapable of degrading dioxin. These transformants are then screenedaccording to the above-described method to identify the gene involved inthe degradation process of dioxin.

The kit of the present invention for screening for an organism or anenzyme capable of degrading dioxin, or a gene encoding an enzyme capableof degrading dioxin, is characterized in that it includes theesculetin-type dioxin analogue of the general formula (3) or thenaphthalene-type dioxin analogue of the general formula (4).

The screening kit is adapted to implement the above-described method forscreening for an organism or an enzyme capable of degrading dioxin or agene encoding an enzyme capable of degrading dioxin and includes anesculetin-type dioxin analogue or a naphthalene-type dioxin analogue ina crystallized form to serve as a substrate, an organic solvent (DMSO oracetone) for dissolving the substrate, and an alkaline buffer (100 mMglycine, 100 mM sodium hydroxide, pH 10) used in the detection of thefluorescence. The kit is used according to the procedure described inthe foregoing paragraph.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

0.19 g (11.0 mM) 4-methylesculetin, 0.072 g (1.8 mM) sodium hydride (60%suspension in oil), and 0.476 g (1.8 mM) crown ether (18-Crown-6) wereadmixed with 4 ml hexamethylphosphoramide (HMPA) at room temperatureunder nitrogen atmosphere. The mixture was thoroughly mixed until thesolutes dissolved completely. Subsequently, 0.108 g (0.5 mM)1,2,4,5-tetrachlorobenzene was added to the mixture and the mixture wasstirred at 50° C. to completely dissolve the solute. After dissolving,the reaction was allowed to proceed for 3 hours at 120° C. The reactionwas constantly monitored by thin-layer chromatography (developingsolvent, ethyl acetate:n-hexane=1:3). Upon completion of the reaction,the reaction mixture was allowed to cool to room temperature and wasdiluted with 50 mL ethyl acetate, followed by washing 3 times withsaturated brine. The organic layer was then concentrated under reducedpressure. The resulting concentrate was purified by silica gel columnchromatography (eluant, ethyl acetate:n-hexane=1:3) to obtain 0.12 g ofthe desired compound as an oily product. The oily product was thencrystallized with chloroform and hexane to give 86 mg of thecrystallized compound of the chemical formula (1). The physicalproperties of the resulting compound are as follows:

mp: 296-298° C. ¹H-NMR (δ: ppm) [CDCl₃] 2.34 (3H, s), 6.19 (1H, s), 6.82(1H, s), 6.96 (1H, s), 6.99 (1H, s), 7.02 (1H, s) ¹³C-NMR (δ: ppm)[CDCl₃] 18.7, 105.1, 111.1, 114.0, 116.3, 117.8, 118.0, 127.1, 127.5,137.9, 139.8, 140.4, 143.9, 150.5, 160.4

EXAMPLE 2

0.16 g (11.0 mM) 2,3-dihydroxynaphthalene, 0.072 g (1.8 mM) sodiumhydride (60% suspension in oil), and 0.476 g (1.8 mM) crown ether(18-Crown-6) were admixed with 4 ml hexamethylphosphoramide (HMPA) atroom temperature under nitrogen atmosphere. The mixture was thoroughlymixed until the solutes dissolved completely. Subsequently, 0.108 g (0.5mM) 1,2,4,5-tetrachlorobenzene was added to the mixture and the mixturewas stirred at 50° C. to completely dissolve the solute. Afterdissolving, the reaction was allowed to proceed for 3 hours at 130° C.The reaction was continuously monitored by thin-layer chromatography(developing solvent, ethyl acetate:n-hexane=1:3). Upon completion of thereaction, the reaction mixture was allowed to cool to room temperatureand was diluted with 50 mL ethyl acetate, followed by washing 3 timeswith saturated brine. The organic layer was then concentrated underreduced pressure. The resulting concentrate was purified by silica gelcolumn chromatography (eluant, ethyl acetate:n-hexane=1:3) to obtain0.098 g of the desired compound as an oily product. The oily product wasthen crystallized with hexane to give 68 mg of the crystallized compoundof the chemical formula (2). The physical properties of the resultingcompound are as follows:

mp: 136-138° C. ¹H-NMR (δ: ppm) [CDCl₃] 7.03 (2H, s), 7.24 (2H, s), 7.34(1H, s), 7.35 (1H, s), 7.63 (1H, s), 7.64 (1H, s) ¹³C-NMR (δ: ppm)[CDCl₃] 112.5, 117.8, 125.7, 126.5, 126.9, 131.0, 140.6, 140.7

EXAMPLE 3

Different species of wood-rotting fungi (i.e., Grammothele fuligo WD844,Phanerochaete crassa WD1694, Phanerochaete chrysosporium ME446,Fomitopsis palustris, Coriolus versicolor, and Pleurotus pulumonairus)were used as test subjects and were screened for the ability to degradethe dioxin analogue obtained in Example 1 (esculetin-type analogue).

Each subject fungal species, which had been pre-cultured on potato agarand stamped out of the agar using a cork borer, was inoculated onto 10ml modified NS medium (3% glucose, 1% peptone, inorganic salts, pH 5.0)in a 100 ml Erlenmeyer flask and was cultured for 5 to 7 days at 28° C.Subsequently, a 100 μl DMSO solution containing the substrate(esculetin-type dioxin analogue obtained in Example 1) at a finalconcentration of 1 ppm was added to the culture. The solution was addedso that the concentration of DMSO in the medium was 1%. Samples of theculture solution were collected at predetermined time intervals (i.e.,1, 3, and 5 days after the addition of the substrate). To a 100 μlportion of each sample, 1.9 ml of an alkaline buffer (100 mM glycine,100 mM sodium hydroxide, pH 10.0) was added. The sample mixtures werethen exposed to long-range UV light (365 nm) as excitation light andwere visually screened for those emitting fluorescence. On day 7, thesamples were irradiated with excitation light of 350 nm and, using afluorescence spectrophotometer, the intensity of fluorescence wasmeasured at 450 nm. The results are shown in Table 1 below. A controlshown in Table 1 is the above-described culture medium with only dioxinanalogue (esculetin-type) added.

TABLE 1 Fluorecsence Subject Fungi intensity Control 1370 G. fuligoWD844 1639 P. crassa WD1694 1419 P. pulumonairus 2203 F. palustris 1314C. versicolor 1601 P. chrysosporium 1619

As shown by the results above, Pleurotus pulumonairus caused the mostsignificant fluorescence emission, followed by WD844 (Grammothelefuligo), ME446 (Phanerochaete chrysosporium), and then Coriolusversicolor. Pleurotus pulumonairus was found to cause more intensefluorescence than did ME446 (Phanerochaete chrysosporium), awood-rotting fungus known for its ability to degrade dioxin.

EXAMPLE 4

Different species of wood-rotting fungi (i.e., Grammothele fuligo WD844,Phanerochaete crassa WD1694, Phanerochaete chrysosporium ME446,Fomitopsis palustris, Coriolus versicolor, and Pleurotus pulumonairus)were used as test subjects and were screened for the ability to degradethe dioxin analogue obtained in Example 2 (naphthalene-type analogue).

Each subject fungal species, which had been pre-cultured on potato agarand stamped out of the agar using a cork borer, was inoculated onto 10ml modified NS medium (3% glucose, 1% peptone, inorganic salts, pH5.0)in a 100 ml Erlenmeyer flask and was cultured for 5 to 7 days at 28° C.Subsequently, a 100 μl DMSO solution containing the substratenaphthalene-type dioxin analogue obtained in Example 2 at a finalconcentration of 1 ppm was added to the culture. The solution was addedso that the concentration of DMSO in the medium was 1%. Samples of theculture solution were collected at predetermined time intervals (i.e.,1, 3, and 5 days after the addition of the substrate). To a 100 μportionof each sample, 1.9 ml of an alkaline buffer (100 mM glycine, 100 mMsodium hydroxide, pH10.0) was added. The sample mixtures were thenexposed to long-range UV light (365 nm) as excitation light and werevisually screened for those emitting fluorescence. As a result, it wasproven that Pleurotus pulumonairus caused the most intense fluorescenceemission than the other species of the wood rotting fungi tested.

REFERENCE EXAMPLE 1

Pleurotus pulumonairus, which has proven to cause the most intensefluorescence in Examples 3 and 4, was examined for its ability todegrade dioxin.

As in Examples 3 and 4, Pleurotus pulumonairus was cultured in a culturemedium containing glucose and peptone and, on day 7,2,3,7,8-tetrachlorodioxin was added to the medium at a finalconcentration of 0.5 ppm. The fungus was subsequently cultured for 20days. After the culturing period, the culture medium containing thefungus was freeze-dried and was subjected to extraction by refluxingwith hexane. Hexane was then removed by distillation and the remainingculture was diluted 200-fold with 200 ml methanol containing 100 ppmTriton X-100. Using a High Performance Dioxin Immunoassay Kit (CapeTechnologies, United States) according to the manufacturer'sinstruction, the concentration of dioxin was measured to determine thedegradation rate of dioxin. The results are shown in Table 2 below. Acontrol shown in Table 2 is the above-described culture medium with only2,3,7,8-tetrachlorodioxin added. Fomitopsis palustris was used as anegative control.

TABLE 2 Fungus Degradation rate Control 0% Pleurotus pulumonairus 31% Fomitopsis palustris 9%

As can be seen from the results of Table 2, Pleurotus pulumonairus,which has proven to cause the most intense fluorescence in Examples 3and 4, showed a significant ability to degrade dioxin. The 9%degradation rate observed for Fomitopsis palustris, which is incapableof degrading dioxin, is considered to have resulted from non-specificbinding of dioxin to the fungal cells, rather than from the degradationof dioxin.

INDUSTRIAL APPLICABILITY

The present invention allows rapid, simple, and highly sensitivescreening for organisms that can degrade, metabolize, and detoxifydioxins rapidly and effectively and can thus be applicable to thebioremediation of the environment. The present invention thereforeenables construction of highly effective bioremediation system thathelps eliminate the problem of dioxin contamination, a problemincreasingly becoming an issue of public concern.

Furthermore, the present invention allows biochemical analysis(enzymatic-chemical, kinetic, or molecular biological analysis) ofdegradative/metabolic systems of dioxins in living organisms, therebymaking it possible to accumulate fundamental data on molecular evolutionrequired for the purposes of developing novel bio-reactors for cleaningthe environment and designing high-performance degradative enzymes byprotein engineering.

1. A novel dioxin analogue which is formula (1):


2. A method for screening for an organism or an enzyme capable ofdegrading dioxin, or a gene encoding an enzyme capable of degradingdioxin, comprising the steps of contacting the dioxin analogue accordingto claim 1 as a substrate in a reaction with a composition comprising acell culture of an organism of interest, an enzyme of interest, or acell culture of a recombinant organism having a gene encoding an enzymeof interest to form a reaction mixture; and detecting the fluorescenceemitted by the fluorescent metabolites resulting from the reaction ofsaid dioxin analogue with said composition.
 3. A method for screeningfor an organism or an enzyme capable of degrading dioxin, or a geneencoding an enzyme capable of degrading dioxin, comprising the steps ofcontacting a dioxin analogue as a substrate in a reaction with acomposition comprising a cell culture of an organism of interest, anenzyme of interest, or a cell culture of a recombinant organism having agene encoding an enzyme of interest to form a reaction mixture; anddetecting the fluorescence emitted by the fluorescent metabolitesresulting from the reaction of said dioxin analogue with saidcomposition, wherein the dioxin analogue is formula (3):

wherein X is a chlorine atom, m is an integer from 1 to 4, and R¹ is ahydrogen atom or a lower alkyl group having 1 to 3 carbon atoms.
 4. Ascreening kit for an organism or an enzyme capable of degrading dioxin,or a gene encoding an enzyme capable of degrading dioxin, the kitcomprising a dioxin analogue which is the following general formula (3):

wherein X is a chlorine atom, m is an integer from 1 to 4, and R¹ is ahydrogen atom or a lower alkyl group having 1 to 3 carbon atoms.